Flat-Field Imaging System and Methods of Use

ABSTRACT

A method of aligning a plurality of targets is provided. The method includes generating a plurality of targets. A third phase includes the plurality of targets. The method further includes combining a first phase, a second phase, and the third phase in a volume. The first phase, the second phase, and the third phase are substantially immiscible, and the third phase is in fluid communication with the first phase and the second phase, and the first phase, the second phase, and the third phase are operable to be in a configuration of the third phase between the first phase and the second phase in the volume.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. provisionalapplication Ser. No. 61/498,440, filed Jun. 17, 2011, which isincorporated herein by reference in its entirety.

BACKGROUND

Generally, there has been an increasing need for effective separation,alignment, and manipulations of colloidal and cellular suspensions ordroplets and other particles based on the increasing number of systemsutilizing microscale transport properties. These types of systems havesignificant parallelization and high throughput. Examples ofapplications for these systems include genetic analysis, molecularseparations, sensors, imaging, printing, and surface patterning.

In one example, manipulation and positioning of the colloidal andcellular suspensions or droplets, and other particles is useful ifimaging of the particles is desired. For example, the use offluorescence detection is a ubiquitous practice in microbiology andbiochemistry as well as colloidal science, biophysics and several otherdisciplines. Labeling cells, cellular components or individualbiomolecules, or particles with molecular or colloidal fluorescentprobes has enabled the visualization of several cellular metabolic andbio-molecular assembly processes. As such, methods involving fluorescenttagging, excitation, and detection may rely on methods of aligning,sorting, and manipulations.

An example of a known separation system is a fluorescence activated cellsorting (FACS) system that sorts and manipulates cells in continuousmicrofluidic flows. Fluorescence labeling of cells combined withtraditional macroscopic FACS systems allow for the identification andseparation of rare cells from concentrated suspensions, thesequestration of cells displaying desired physiological properties ormetabolic states, and the parsing of large combinatorial libraries forspecific information. A FACS system, however, can be complex andcumbersome. Furthermore, FACS, as well as other known alignment andsorting methods, may be improved by simplifying signal acquisition andinterpretation to allow for closer to real-time feedback.

SUMMARY

In one exemplary embodiment, a method of aligning a plurality of targetsis provided. The method may include generating a plurality of targets. Athird phase may include the plurality of targets. The method may furtherinclude combining a first phase, a second phase, and the third phase ina volume. In some embodiments, the first phase, the second phase, andthe third phase may be substantially immiscible, and the third phase maybe in fluid communication with the first phase and the second phase, andthe first phase, the second phase, and the third phase are operable tobe in a configuration of the third phase between the first phase and thesecond phase in the volume.

BRIEF DESCRIPTION OF THE FIGURES

A better understanding of the features and advantages of the presentdisclosure will be obtained by reference to the accompanying drawings,which are intended to illustrate, not limit, the present teachings.

FIG. 1 is an exemplary flowchart of a method for positioning a pluralityof targets according to various embodiments of the present teachings;

FIG. 2 is an exemplary flowchart of a method for positioning a pluralityof targets according to various embodiments of the present teachings;

FIG. 3 illustrates a cross-section of a volume for aligning a pluralityof targets according to various embodiments of the present teachings;

FIG. 4 illustrates an imaging system according to various embodiments ofthe present teachings;

FIG. 5 is an exemplary flowchart of a method for forming targetsaccording to various embodiments of the present teachings;

FIGS. 6A, 6B, and 6C illustrate various configurations of volumes forpositioning a plurality of targets according to various embodiments ofthe present teachings;

FIGS. 7A, 7B, and 7C illustrate various configurations of volumes forpositioning a plurality of targets according to various embodiments ofthe present teachings;

FIG. 8 illustrates an exemplary image taken of the positioned pluralityof targets according to various embodiments of the present teachings;and

FIG. 9 illustrates another exemplary image taken of the positionedplurality of targets according to various embodiments of the presentteachings.

DETAILED DESCRIPTION

To provide a more thorough understanding of the present invention, thefollowing description sets forth numerous specific details, such asspecific configurations, parameters, examples, and the like. It shouldbe recognized, however, that such description is not intended as alimitation on the scope of the present invention, but is intended toprovide a better description of the exemplary embodiments.

The present application relates to methods and systems for aligning andpositioning desired targets in fluid. Aligning or sorting desiredsamples, particles, objects or other targets in a fluid has been achallenge. Often, the desired targets are included in a large volumemaking efficient and fast extraction or analysis, for example,difficult. According to various embodiments described herein, methodsand systems of aligning a plurality of targets in a flat-fieldconfiguration for extraction, sorting, or imaging in a single field ofview are provided.

The generation of the plurality of targets and examples of applicationsusing a flat-field imaging system are described in provisionalapplications 61/470,713, filed on Apr. 1, 2011, and 61/481,085, filed onApr. 29, 2011, both entitled System And Method For DeterminingCopies-Per-Unit-Volume Using PCR And Flow Control Of Droplets, and bothof which are incorporated herein by reference in their entirety.

FIG. 1 illustrates a method of aligning a plurality of targets in fluidaccording to various embodiments described herein. In step 102, aplurality of targets is generated. The plurality of targets may beincluded in a third phase. The plurality of targets may be an emulsion,porous beads, or hollow beads, among other things for example. In someembodiments, the plurality of targets may contain a biological sample.

In various embodiments, the plurality of targets may initially be in theother phases. However, after some settlement of the at least threephases, the plurality of targets will congregate within the third phase.Furthermore, in some embodiments, the plurality of targets may have asimilar density as the first or second phases. In yet other embodiments,the plurality of targets may have densities similar to the first, secondand third phase. In these situations, sorting of the plurality oftargets may be possible since the targets with densities correspondingto the density of a phase will eventually settle within that phase.

In step 104, a first phase, a second phase, and the third phase,including the plurality of targets, are combined in a volume. Accordingto embodiments described herein, a volume may be a volume of phases. Aphase may be a fluid, such as a liquid, that surrounds the targets. Invarious embodiments, the volume may include two or more differentphases.

The first phase, the second phase, and the third phase are substantiallyimmiscible with each other. According to embodiments described herein,the two or more phases, or fluids, may have different densities,viscosities, interfacial tension, laminar flow for a flowing system, orany other suitable property which prevents two adjacent liquids frommixing. In embodiments of the teachings described herein, substantiallyimmiscible means up to 50% of a phase remains unmixed with anotherphase. In other words, substantially immiscible also is defined as up to50% of the phase may be mixed with another phase. The amount of a phasethat mixes with another phase may depend on the density of the phases,for example, as well as other characteristics. In other embodiments, thevolume may include a fluid having a non-uniform density.

Further, within the volume, the third phase is in fluid communicationwith the first phase and the second phase. The third phase, includingthe plurality of targets, is operable to be positioned between the firstphase and the second phase. As such, the plurality of targets is withinthe layer of the third phase between the first phase and the secondphase. In this way, the targets are positioned so they are groupedtogether within the volume. A volume according to various embodiments isillustrated in FIG. 3 and will be described in more detail below.

In some embodiments, the plurality of targets may be positioned to be ina flat-field configuration so that the targets are within the field ofview of an optical sensor, as described in more detail below. An opticalsensor may be a camera, such as a CMOS or CCD camera, PMT, or any otheroptical sensing technology, for example. The optical sensor may be in ascanning flat-bed configuration, for example. In other embodiments, theplurality of targets are sorted or extracted in a more rapid manner thanprevious methods.

Imaging

To improve accuracy and quality of an image, the targets desired to beimaged should be in the field of view of the optical sensor.Furthermore, a more successful image of targets in a fluid will have asmany of the targets in focus and have as very little overlap of thetargets in the image. Thus, the improved systems and methods foraligning and positioning targets described herein may be used for animproved imaging system.

According to embodiments described herein, targets are objects withinthe imaging volume that are desired to be imaged. Targets may bedroplets, hollow beads, magnetic beads, or any other object which isdesired to be imaged. A goal for imaging the targets, for example, is tooptically measure properties of the targets. Properties that may bemeasured from the image are the size and/or volume of the targets,fluorescence emissions from the target either at a single wavelength ormultiple wavelengths, turbidity, optical density, or any other suitabledetection characteristic, for example.

FIG. 2 is a flowchart illustrating an exemplary method according toembodiments of the present teachings. The method includes, in step 202,forming a volume. The volume includes a first phase, a second phase, anda third phase. In various embodiments, the first phase and second phaseare different fluids, and the third phase comprises the targets. Thesethree phases are substantially immiscible. The third phase may haveproperties such that it is positioned between the first phase and thesecond phase. The third phase may then be within a field of view of anoptical sensor. The method further may include, in step 204, imaging theplurality of targets. In other words, the third phase, including thetargets, may be positioned above the first phase and below the secondphase. Further, the targets may be imaged in focus since the targets arewithin the field of view of a camera.

The side-view cross-section of an exemplary volume is illustrated inFIG. 3. A first phase 301 is depicted below a second phase 302. Betweenthe first phase 301 and the second phase 302, is a third phasecomprising targets 303. Here, the first phase 301 has a higher densitythan both the second phase 302 and third phase, including targets 303.The third phase, including the targets 303, has a higher density thanthe second phase 302. The volume may be contained in vessel 306.

In certain embodiments, the viscosity of phases affects the stability ofthe targets in the third phase. For example, a second phase that is moreviscous than the first phase may improve the stability of the pluralityof targets within the third phase.

In some embodiments, the third phase 303 may include the targets. Inthis configuration shown in FIG. 3 according to various embodiments, thetargets are captured in a location between the first phase 301 and thesecond phase 302. The first phase 301 may be a liquid such as afluorinated fluid (HFE), or a mixture of different fluorinated fluids,for example. The second phase 302 maybe a mineral oil, for example.Mineral oil does not dissolve in a fluorinated fluid.

Targets 303 of the third phase may be, but are not limited to, droplets,and non-magnetic beads, including porous beads, or hollow beads, forexample. The droplets may be an emulsion. The porous or hollow beads maybe spherical or cylindrical. The targets may comprise a passivereference dye, a light-scattering enhancing material, q-dots, a protein,a colloidal metal, colloidal gold, a reporter dye, areaction-independent flow marker, or a combination thereof. In someembodiments, each of the plurality of targets is a discrete sampleportion. The plurality of targets may comprise a primer pair, anucleotide probe, a Taq polymerase, or a single cell, among other thingsor any other suitable particle for capture in a droplet, for example.

Second phase 302 prevents evaporation of the third phase and the firstphase 301. The second phase 302 on top of the targets may help to reducebreakage of the droplets, if the droplets are an emulsion according tosome embodiments. Furthermore, second phase 302 may help reduce theoverlapping of the targets 303 between the first phase 301 and thesecond phase 302. Second phase 302 may also help reduce the motion ofthe droplets because of the viscosity of the second phase.

An exemplary imaging system is depicted in FIG. 4. The volume, as inFIG. 3, comprises a first phase 301 on the bottom of the volume. A thirdphase including targets 303 is held in between a top second phase 302.As such, the targets 303 are in a substantially flattened configuration.Targets are configured such that they are within the field of view, orthe focal plane, of an optical sensor 402. As mentioned above, anoptical sensor may be a camera, such as a CMOS or CCD camera, PMT, orany other optical sensing technology, for example. The optical sensormay be in a scanning flat-bed configuration, for example. Any standardlight source may also be used in the imaging system. For example, thelight source may be a laser or LED, in some embodiments. The lightingconfiguration may also be reflective or passthrough. The depth of focusincludes the targets 303. The image taken with optical sensor 402 mayinclude most or all of the targets 303 in focus so that a more completeand accurate analysis may be performed. The optical sensor may bepositioned above or below the volume to image the plurality of targets.In other embodiments, the optical sensor may be a scanning opticalsensor, similar to a flatbed scanning system.

Examples of Applications

In various embodiments, images taken in accordance with the embodimentsdescribed herein may be analyzed to identify and quantify individualproteins, nucleic acids, or other species that constitute the solutes.The flat-field configuration for a plurality of targets according toembodiments described in this document may be used in applicationsinvolving image analysis, extraction, or sorting of targets, forexample, but are not limited to these applications.

For example, in various embodiments, the methods and systems describedherein may be used to detect other biological components of interest.These biological components of interest may be any suitable biologicaltarget including, but are not limited to, DNA sequences (includingcell-free DNA), RNA sequences, genes, oligonucleotides, molecules,proteins, biomarkers, cells (e.g., circulating tumor cells), or anyother suitable target biomolecule.

In various embodiments, such biological components may be used inconjunction with various PCR, qPCR, and/or dPCR methods and systems inapplications such as fetal diagnostics, multiplex dPCR, viral detectionand quantification standards, genotyping, sequencing validation,mutation detection, detection of genetically modified organisms, rareallele detection, and copy number variation.

Furthermore, as used herein, thermal cycling may include using a thermalcycler, isothermal amplification, thermal convention, infrared mediatedthermal cycling, or helicase dependent amplification, for example. Insome embodiments, the chip may be integrated with a built-in heatingelement.

According to various embodiments, detection of a target may be, but isnot limited to, fluorescence detection, detection of positive ornegative ions, pH detection, voltage detection, or current detection,alone or in combination, for example.

In one example, a plurality of targets according to various embodimentsmay be used in digital Polymerase Chain Reaction (dPCR). DPCR is amethod that has been described, for example, in U.S. Pat. No. 6,143,496to Brown et al. Results from dPCR can be used to detect and quantify theconcentration of rare alleles, to provide absolute quantitation ofnucleic acid samples, and to measure low fold-changes in nucleic acidconcentration.

dPCR is often performed using an apparatus adapted from conventionalqPCR, in which replicates are arrayed in a two dimensional array formatincluding m rows by n columns, i.e., an m×n format. PCR cycling andread-out (end-point or real-time) generally occurs within the samearray. A maximum of m×n replicates can be processed in a single batchrun. Generally, increasing the number of replicates increases theaccuracy, precision, and reproducibility of dPCR results.

The (m×n) format in most quantitative polymerase chain reaction (qPCR)platforms is designed for sample-by-assay experiments, in which PCRresults need to be addressable for post-run analysis. For dPCR, however,the specific position or well of each PCR result may be immaterial andonly the number of positive and negative replicates per sample may beanalyzed.

The read-out of dPCR, that is, the number of positive reactions and thenumber of negative reactions, may be used to calculate starting templateconcentration based on a Poisson equation, as follows:

${f\left( {k;\lambda} \right)} = {\frac{\lambda^{k}^{- \lambda}}{k\; l}.}$

On the other hand, the read-out of qPCR (signal vs. cycle) isproportional to the log of the template concentration. For this reason,dPCR typically is constrained to a narrow dynamic range of templateinput.

According to various embodiments, a dPCR analysis of a sample mayinclude preparing and analyzing uniform or variously-sized targets.These targets may be sample portions, such as poly dispersed ormulti-mono dispersed emulsions.

Multi-mono dispersed emulsions, also referred to as polydispersedemulsions, are less difficult to make, minimally handled, can be formedin batches, and greatly increase the dynamic range of dPCR. Furthermore,a small reaction chambers may allow analysis without sample dilutionthat can introduce error. For example, heat, shaking, sonic energy,ultrasonic baths, combinations thereof, and the like can be used toproduce emulsions, for example, to process batches of emulsions in96-well, 384-well plates, or cell culture plates without the need forany special consumables to physically touch the samples. In otherembodiments, a plate may be used based on the amplification apparatus.This greatly reduces the chance of cross-contamination. In addition,multi-mono dispersed emulsions may typically vary in volume on the orderof 1 fL to 50 μL. In some embodiments, multi-mono dispersed emulsionsmay vary in volume from about 1 pL to 500 pL, eliminating the need todilute samples to achieve terminal dilutions.

As mentioned above, multi-mono dispersed emulsions may be imaged andanalyzed according to various embodiments. A multi-mono dispersedemulsion may include two or more sizes of targets, where the sizes areknown or predetermined. For example, a multi-mono dispersed emulsion maycontain three different sizes of targets that are substantially the samesize as three different predetermined sizes. Substantially the same sizemeans within +/−10% of the predetermined size. By determining which sizeof the different predetermined sizes each discrete sample portion is,the volume of each discrete sample portion can then be determined.Multi-mono dispersed emulsions may vary in volume from about 1 fL to 10pL. In other words, each droplet can be binned into a predeterminedsize. In this way, the dynamic range can be increased and analysis of animage may be simplified.

The sample portions are amplified so that the sample portions containthe target nucleic acid. Amplification may be performed by polymerasechain reactions (PCR) with target concentration near terminal dilution.The volume of the sample portions may be known. If the sample portionsare different sizes, the volume of the sample portions may need to bedetermined. The positive and negative reactions within the plurality ofsample portions are counted. More particularly, the number of sampleportions that contain successful amplification of the target nucleicacid are counted. The sizes and the positive and negative reactions maybe determined by imaging the sample portions, as targets, according toembodiments of the present teachings, for example. The average copynumber per reaction is estimated. The estimation may be made using aPoisson distribution. Then, the target copy number per unit volume inthe starting sample is estimated.

As described above, an image generated by various embodiments may beused to estimate the volume of the plurality of targets, for dPCRanalysis.

The image generating according to various embodiments may be used tocompare the plurality of amplified sample portions, the targets, to aplurality of standards of known respective volumes, for example, aplurality of standards of known respective volume that uniformly sizedor that are of different known volumes. Analysis of the image mayfurther comprise subjecting a plurality of portions of a standard to thesame nucleic acid amplification conditions to form a plurality ofprocessed standards, wherein each of the processed standards are of aknown respective volume, and then comparing the image of the pluralityof processed standards to the plurality of processed sample portions. Insome embodiments, the plurality of sample portions have an average offrom about 0.1 to about 0.8 copy of the target nucleic acid per discreteloaded mixture. The plurality of sample portions may have an averagediameter of from about 0.3 micrometer (μm) to about 600 μm, or anaverage diameter of from about 1.0 μm to about 100 μm, or an averagevolume of from about 0.5 femtoliter (fL) to about 1 microliter (μL), oran average volume of from about 10.0 fL to about 100 nanoliters (nL).However, in some embodiments, a sample portion may be as large as 65 pL.

In an example of image analysis of an image generated by embodiments ofthe present teachings, sample portions of first, second, and thirdvolumes of different known respective standard sizes may contain first,second, and third respective detectably unique dyes and may beidentified and used to scale the size of the discrete sample portionshaving unknown volume sizes. Sample portion may be generated with sizesof from about 0.3 μm in diameter up to about 1000 μm in diameter, forexample, from about 0.4 μm in diameter up to about 300 μm in diameter,from about 0.5 μm in diameter up to about 200 μm in diameter, or fromabout 1.0 μm in diameter up to about 100 μm in diameter. Sample portionvolumes are of up to about 1.0 μL in size may be produced and processed.Sample portion volumes based on spherical diameters measured throughimage analysis can be estimated, for example, using a conversion chartsuch as this one:

Radius diameter volume 0.6 uM 1.2 uM 1 fL e. coli 1.4 uM 2.8 uM 10 fL 3uM 6 uM 100 fL 6 uM 12 uM 1 pL 14 uM 28 uM 10 pL human cell 30 uM 60 uM100 pL 60 uM 120 uM 1 nL 140 uM 280 uM 10 nL 300 uM 600 uM 100 nL 600 uM1200 uM 1 uL

Measuring the size of each of the plurality of processed sample portionsmay comprise analyzing each of the plurality of processed sampleportions, and the analyzing may comprise one or more of measuring oranalyzing an index of refraction, a light scattering property, a forwardlight scattering property, a side light scattering property, an opticalabsorption property, an optical transmission property, a peak height ofan optical signal, a peak width of an optical signal, a fluorescentproperty, a time-of-flight fluorescent property, or a combinationthereof. The method may further comprise estimating what size ofprocessed sample portion provides a specific percentage of processedsample portions of that size that test positive for the presence of oneor more of the at least one target nucleic acid, or estimating what sizeprocessed sample portion of the differently-sized processed sampleportions provides a 50% positivity rate with regard to determining thepresence of one or more of the at least one target nucleic acid.

In various embodiments, the methods and systems described herein may beused to detect other biological components of interest. These biologicalcomponents of interest may include, but are not limited to, cells andcirculating tumor cells, for example. Furthermore, in addition to dPCR,the methods and systems in various embodiments may be used inapplications, such as fetal diagnostics, multiplex DPCR, viraldetection, genotyping, and rare allele detection copy number variation.

Generation of the Plurality of Targets

According to various embodiments, the targets may also be generated. Anemulsion apparatus may generate a plurality of targets. The emulsionapparatus may generate the plurality of targets by various methods, suchas shaking, stirring, sonicating, extruding, or electrowetting, forexample. In some embodiments, the emulsion apparatus may be a sonicator,a vortexer, or a plate shaker. In other embodiments, magnetic beads maybe used to stir a third phase to generate the plurality of targets.Emulsification parameters, such as emulsification method,strength/power, time, oil/surfactant chemistry, viscosity,concentration, aqueous phase composition, and water-to-oil ratio, forexample, may be optimized to produce desired sizes for the targets. Insome embodiments, the targets have a diameter of between 10μ, to 150 μmand a volume of between 1 pL to 1 nL.

Exemplary systems for methods of preparing and processing emulsions thatmay be used according to the present teachings include those describedin U.S. patent application Ser. No. 12/756,547, filed Apr. 8, 2010, toLau et al. for “System and method for preparing and using bulkemulsion,” which is incorporated herein in its entirety by reference.Exemplary systems for methods of processing and thermally cyclingemulsions that may be used according to the present teachings includethose described in U.S. patent application Ser. No. 12/756,783, filedApr. 8, 2010, to Liu et al. for “System comprising dual-sided thermalcycler and emulsion PCR in a pouch,” which is also incorporated hereinin its entirety by reference.

Generating the targets may also include diluting the sample to form adiluted sample and forming the plurality of targets from the dilutedsample. Dilution may comprise terminally diluting the sample to achievean average of less than one of the at least one target nucleic acidmolecules per imaging target. In some embodiments, the method furthercomprises: serially diluting different portions of the sample bydifferent respective dilution ratios; dividing each serially dilutedportion into a plurality of aliquots; and processing each of theplurality of aliquots of each of the serially diluted portions.

According to various embodiments, the components of the plurality oftargets may be provided in a multi-well plate. Forming the plurality oftargets may include emulsifying an aqueous sample with a medium that isat least substantially immiscible with the sample. In some embodiments,the emulsifying may comprise mixing the aqueous sample with the mediumthat is at least substantially immiscible with the sample in themulti-well plate, sonicating the aqueous sample with the medium that isat least substantially immiscible with the sample in the multi-wellplate, shaking the aqueous sample in the medium that is at leastsubstantially immiscible with the sample in the multi-well plate, orstirring the aqueous sample in the medium that is at least substantiallyimmiscible with the sample in the multi-well plate. According to variousembodiments, surfactant may be added to a phase to generate theplurality of targets, or discrete sample portions. The quality of theplurality of targets that are generated may depend on the components ofthe phases. For example, the second phase containing a surfactant maymake a plurality of targets with good integrity in the third phaseincluding certain components.

According to various embodiments, imaging target stability is a relevantfactor. Stability ensures that the targets when in proximity to eachother, the targets do not coalesce. Very stable emulsions allow veryhigh density of targets since targets can “touch” each other whilemaintaining a minimal layer of oil in between. As such, emulsificationto form a plurality of targets may also take place in the presence of asurfactant, so that the kinetic stability of the emulsion increases.These surfactants then line the surface of the targets, stabilizing it.Some surfactants which may be used, but are not limited to, are Span80,STF9, ABIL EM90, KRYTOX, and DC BY11-030, for example.

Any of a variety of substantially or totally immiscible fluids can beused as the carrier fluid. Immiscibility is determined with respect tothe aqueous sample droplets, the targets. The substantially or totallyimmiscible fluid may comprise, for example, paraffin oil, mineral oil,silicone oil, a perfluorinated polyether (PFPE), other fluorinatedfluids, fluorinated solvents, combinations thereof, and the like. Somespecific fluids that can be used as a carrier fluids include GALDEN®HT170 available from SOLVAY SOLEXIS of West Deptford, N.J., otherGALDEN® HT liquids available from SOLVAY SOLEXIS, FC-40 available from3M Company of St. Paul, Minn., and other FLUORINERT™ liquids availablefrom 3M Company of St. Paul, Minn.

Generating Targets in the Presence of the First and Second Phases

According to various embodiments, emulsification to form the pluralityof targets may be in the presence of the first phase and the secondphase. In some embodiments of the present teachings, the plurality oftargets may be generated in the volume in the presence of the first andsecond phases. In other embodiments, the plurality of targets may begenerated outside the volume in a vessel and added to the volume alreadycontaining the first phase and the second phase. An exemplary method toform the targets in the presence of the first phase and the second phaseis shown in FIG. 5.

In step 502, a first phase, a second phase, and a third phase are addedtogether to form a mixture. The first phase, the second phase, and thirdphase are substantially immiscible. The mixture may contain surfactant.The third phase may comprise, in certain embodiments, the sample asdescribed above. The mixture is agitated in step 504 to form a pluralityof targets. The agitation may be from shaking, stirring, sonicating,extruding, or electowetting, for example. In general, there is arelationship between the energy of agitation and the size distributionof the plurality of targets. For example, higher energy agitation maygenerate smaller-sized targets. As described above, the targets may bedroplets of various sizes, a multi-mono dispersed emulsion(polydispersed emulsion), or of two or more known sizes, a multi-monodispersed emulsion. In step 506, the mixture is allowed to rest andsettle so that the three phases separate, according to densities of thephases, to form an imaging volume. The targets settle into positionbetween the first phase and the second phase.

Meniscus of the Third Phase

As mentioned above, the volume may be contained in a vessel 306. Inorder to improve an image taken according to embodiments of the presentteachings, the meniscus of the first phase and second phase may beconsidered. A meniscus may cause the third phase including the targetsto be convex or concave with respect to the optical sensor and affectthe quality of an image.

An exemplary imaging volume 602 is depicted in FIG. 6A. The interfacebetween the phases is flat. In this case, the targets are dispersedsubstantially evenly in the field of view of the optical sensor. Thegenerated image will have more targets in better focus and preventoverlapping of the targets so that more targets may be imaged foranalysis.

FIG. 6B illustrates another exemplary imaging volume 604 with a meniscusthat creates a concave third phase with respect to an optical sensorpositioned above the imaging volume 604. In this case, the meniscuscould force aggregation of targets toward the center of the third phaselayer to a point when the targets overlap. Also, because of the concaveshape of the third phase, the depth of field may not be wide enough toimage the targets in focus at all points. On the other hand, convexmeniscus, with respect to a above-positioned optical sensor, asillustrated in FIG. 6C, may cause targets to be dragged to the edge ofthe vessel 306 and away from the center, which would result in reducednumber of accurately imaged targets.

Thus, according to various embodiments, vessel walls may be coated witha material to modify the meniscus. The coating may include a variety ofhydrophobic materials. For example, the interior walls of the vessel306, containing an imaging volume, may be coated with Teflon. In otherembodiments, the shape of the vessel well may modify the meniscus angle.For example, the vessel 306 may have a well in a cone or inverted coneshape that would flatten out the meniscus angle. In other embodiments,the viscosity of the phases may be considered for minimizing themeniscus angle.

In other embodiments, convex meniscus may be tolerated by overloading orpreloading the meniscus with excess targets. These excess targets maynot be of interest for analyzing. In other words, there may not be anysample within the targets that need to be analyzed by imaging, forexample. In this way, overlapping of targets of interest, i.e., targetscontaining sample, will be minimized and analysis may be more accurate.

Furthermore, in other embodiments, viscosity of the first phase or thesecond phase may alleviate some issues related to the meniscus describedabove. More viscous oils may be used, in some embodiments, to limittarget movement and limit target migration towards the edges of thevessel.

However, in some embodiments, the meniscus allows the smaller targets togather near the meniscus, while the larger targets remain closer to thecenter of the third phase. As such, within the third phase, there may besorting of the smaller-sized targets because the larger-sized targetswould stay closer to the middle of the third phase while thesmaller-sized targets move toward the vessel wall and meniscus. In someembodiments, magnetic beads may be in the third phase and be used toagitate the third phase to assist with the sorting process.

Overlapping of Targets

As described above, multi-mono dispersed emulsions may comprise targetsof a plurality of sizes. In the embodiment depicted in FIG. 7A, thetargets 303 are positioned between the first phase 301 and the secondphase 302 such that the targets 303 have minimal overlap. However, asillustrated in FIG. 7B, there is overlap in targets 303. In particular,smaller targets may be covered by an edge of a larger imaging target. Inanother example, targets 303 may have a density closer to the firstphase 301, pulling larger imaging droplets lower. In this case, targetsmay not be accurately imaged.

In various embodiments, overlapping may be reduced by forming a volumein which the third phase has a density of about the average of thedensities of the first phase and the second phase.

In other embodiments, the overlapping may be reduced by forming a volumewith a first phase, second phase, and third phase, having densities thatare very close. In yet other embodiments, overlapping may be alleviatedby the first and second phases having densities far from the density ofthe third phase.

In some embodiments, the third phase including the targets is diluted.In this way, the targets are spread out enough in the focal layer thatthe overlap events are more infrequent.

In yet other embodiments, there may be a small volume of the first phasein the vessel. In other words, the depth of the first phase in thevessel may be small enough that the larger targets, included in thethird phase, may rest on the bottom of the vessel. As such, the frictionbetween the targets that contact the bottom surface of the vessel maytend to not have as much motion. Thus, this may provide more stabilityto the planar flat-field configuration in the third phase. Additionally,since the larger targets may tend to have less movement than the smallertargets not in contact with the bottom surface of the vessel, this maybe useful for sorting the plurality of targets by size.

On the other hand, in mono dispersed emulsions, when the targets are allsubstantially the same size, there may be no physical space for thetargets to overlap with each other.

FIG. 8 illustrates an exemplary image of a plurality of targets taken inaccordance with embodiments and methods described herein. The imageshown in FIG. 8 illustrates a white light image taken of a plurality oftargets.

FIG. 9 illustrates another exemplary image of a plurality of targetstaken in accordance with embodiments and methods described herein. Inthis example, the targets are droplets containing samples amplified byPCR. Successful PCR reactions can be identified in this image from thefluorescent emissions from some of droplets of the plurality of dropletsimaged.

Although the present invention has been described with respect tocertain exemplary embodiments, examples, and applications, it will beapparent to those skilled in the art that various modifications andchanges may be made without departing from the invention.

1-19. (canceled)
 20. A method for imaging, the method comprising:forming a volume, wherein the volume comprises: a first phase, a secondphase, and a third phase, wherein the first phase, the second phase, andthe third phase are substantially immiscible, the third phase is influid communication with the first phase and the second phase and withinthe field of view of an optical sensor, and the third phase includes aplurality of targets; and imaging, by the optical sensor, the pluralityof targets.
 21. (canceled)
 22. (canceled)
 23. The method of claim 20,wherein the first phase has a first density, the second phase has asecond density, and the third phase as a third density, wherein firstdensity is heavier than the third density and the third density isheavier than the second density.
 24. The method of claim 23, wherein thefirst phase is positioned below the third phase and the third phase ispositioned below the second phase.
 25. The method of claim 23, whereinthe plurality of targets have substantially the same density as thethird density.
 26. The method of claim 20, wherein the first phasecomprises a fluorinated fluid (HFE), and the second phase comprises amineral oil.
 27. The method of claim 20, wherein the plurality oftargets comprises targets of a plurality of sizes. 28-36. (canceled) 37.A system for imaging, the apparatus comprising: a volume, wherein thevolume comprises: a first phase, a second phase, and a third phase,wherein the first phase, the second phase, and the third phase aresubstantially immiscible, the third phase is configured to be positionedbetween the first phase and the second phase, and the third phaseincludes a plurality of targets; and an optical sensor configured tohave a field of view including the third phase for imaging the targets.38. The system of claim 37, wherein the imaging volume is contained in avessel.
 39. The system of claim 37, wherein the optical sensor ispositioned above the imaging volume.
 40. The system of claim 37, whereinthe optical sensor is positioned below the imaging volume.
 41. Thesystem of claim 37, wherein the first phase has a first density, thesecond phase has a second density, and the third phase as a thirddensity, wherein first density is heavier than the third density and thethird density is heavier than the second density.
 42. The system ofclaim 41, wherein the first phase is positioned below the third phaseand the third phase is positioned below the second phase.
 43. The systemof claim 41, wherein the plurality of targets have substantially thesame density as the third density.
 44. The system of claim 37, whereinthe second phase comprises a fluorinated fluid (HFE), and the firstphase comprises a mineral oil.
 45. The system of claim 37, wherein theplurality of targets comprises targets of a plurality of sizes.
 46. Thesystem of claim 37, wherein the third phase further includes a mediumthat is at least substantially immiscible with the plurality of targets.47. The system of claim 46, wherein the medium that is substantiallyimmiscible with the plurality of targets comprises at least one selectedfrom the group consisting of: a mineral oil, a silicone oil, a paraffinoil, a fluorinated fluid, a perfluorinated polyether.
 48. The system ofclaim 37, wherein the plurality of targets comprise a plurality ofdroplets.
 49. The system of claim 37, wherein the plurality of targetscomprises porous beads.
 50. The system of claim 37, wherein theplurality of targets comprises magnetic beads.
 51. (canceled) 52.(canceled)